Enzymatic débridement of necrotic tissue involves the removal of such tissue by means of the action of non-toxic enzymes that are able to degrade denaturated collagen, fibrin and elastin present in the devitalised tissue, preserving the viable tissue.
This technique is preferable to mechanical/surgical débridement because it is more selective towards granulation tissue and is especially suitable in non-infected lesions such as skin ulcers of diverse etiology and of varying depth.
The enzymatic formulations available on the market today contain the proteolytic enzyme collagenase (Noruxol® and Iruxol®) or are constituted by an association of fibrinolysin and desoxyribonuclease (Elase®), all of which are effective (albeit with different results) in removing necrotic tissue, purulent exudate and fibrin (Mekkes J. R., Arch Dermatol Rese, 1998, 290:152-157).
Of great interest from the point of view of application are collagenases produced from bacteria of the Clostridium species, although collagenase produced from Vibrio Alginolyticus (Achromobacter collagenase EC 3.4.24.08; Borivoj k., Matrix Supplement, 1992, 1:127-133; EP0115974 B1) produced from a non-pathogenic strain with a specific activity that is markedly superior to that of the enzyme produced from Clostridium, has been widely described and characterised (albeit not yet marketed).
Collagenase is a protein that is very unstable in aqueous solutions even at low temperatures. Moreover, it can easily be denaturated by chelating agents and various metal ions that can interact with the calcium ion that is essential for the enzymatic activity of the molecule.
It is an enzyme that is extremely sensitive to chemical-physical procedures such as freezing, thawing, freeze-drying and drying, processes that are often necessary in the course of preparing final pharmaceutical formulations. Various different formulations have therefore been tested to find a final composition that contains a stable, and therefore active, collagenase (EP0543521B1, patent application WO96/41870, WO93/00807, WO94/24273).
Albeit still at an experimental stage, the enzyme is used in injectable form to treat Dupuytren's contracture, a deforming condition of the fingers, while the use of collagenase is particularly important in reducing glaucoma, a disorder that causes excessive pressure in the eye with possible damage to the optic nerve, linked with an abnormal deposit of collagen within the duct that drains biological fluids from the anterior chamber of the eye.
The collagenase enzyme is mainly indicated in débridement of burns of varying depth, pressure sores, vascular ulcers and diabetic foot ulcers. Moreover, it is used to treat hypertrophic and keloid scars.
Proper wound healing requires a proper reepithelialisation phase, which follows the phase involving removing the eschar, possibly by surgical and/or enzymatic débridement. Collagen is the chief component of necrotic tissue and, consequently, it is fundamentally important to remove it in order to favour reepithelialisation of the wound. However, in the course of this operation it is necessary to protect the surrounding skin to avoid painful irritative phenomena resulting from the use of collagenase, as the enzyme is capable of degrading both denaturated and natural collagen, hydrolysing the peptide bonds of the amino acid chain.
During enzymatic débridement the wound area being treated does not decrease in size, it may even increase. Therefore, when the eschar has been removed, the newly-forming granulation tissue is exposed and consequently open to dangerous bacterial infections that may jeopardise complete healing of the lesion.
The wound healing process is a complex phenomenon involving many types of cellular and humoral factors, and many phases that can favour the formation of pathological scarring, such as hypertrophic and keloid scars.
Proper healing therefore requires the application of drugs to guide (and accelerate) the wound healing process.
Scientific and patent literature contains ample descriptions and claims naming hyaluronic acid (HA) as the chief factor in the tissue regeneration processes (European patent application EP1196179).
Indeed, thanks to its special chemical-physical and biological features, hyaluronic acid participates in and modulates all the main sequential phases of wound healing:                inflammation;        formation of granulation tissue;        reepithelialisation;        scar remodelling.        
Owing to its chemical-physical properties, said polysaccharide controls tissue hydration, creating the correct microclimate necessary to fast healing; moreover, its high viscosity protects the wound from possible bacterial and/or viral infections.
Thanks to its biological properties, hyaluronic acid has proved effective in protecting against free radicals, in controlling inflammatory processes and stimulating angiogenesis. Its role in controlling the expression of cytokines and trophic factors has been demonstrated, and in stabilising the granulation tissue by favouring and regulating the flow of fibroblasts and endothelial cells into the wound during reepithelialisation.
Lastly, experimental data have demonstrated an involvement of hyaluronic acid in controlling keratinocyte proliferation and the deposit of collagen in the wound, thus reducing the formation of fibrotic tissue and, therefore, of pathological scarring (John Chen W. Y. et al., Wound Repair and Regeneration, 1999, 7:79-89).
HA is a hetero-polysaccharide composed of alternating residues of D-glucuronic acid and N-acetyl-D-glucosamine. It is a straight-chained polymer with a molecular weight in the range of 50,000 to 13×106 Da, according to the source from which it is obtained and the methods used to prepare it.
It is present in nature in the pericellular gels, the fundamental substance of the connective tissue in vertebrate organisms (of which it represents one of the chief components), in the synovial fluid of joints, in the vitreous humor and umbilical cord.
HA therefore plays an important role in the biological organism (besides those described above), as a mechanical support for the cells of many tissues, such as the skin, tendons, muscles and cartilage.
Said polysaccharide is known to be used as a vehicle for drugs of various kinds, in simple associations or salified with hyaluronic acid, since its special properties of biocompatibility, biodegradability, non-immunogenicity, viscosity and hydratability make it particularly suitable as a release system for drugs and molecules both at a topical and systemic level (EP0197718B1, EPO445255B1).
Indeed, preclinical experiments with HA associated with anti-inflammatories (such as Diclofenac) for topical use have demonstrated that HA significantly increases (compared to controls) absorption of the drug into the skin where, thanks to the specific action of hyaluronic acid, it is compartmented to form a “reservoir”, minimising further absorption through the skin. The drug's action (and efficacy) is thus significantly increased (Brown M. B. et al., JEADV, 2005, 19:309-318).
The Applicant, contrary to the above description of HA as a delivery system, has surprisingly discovered that an association between HA and/or the derivatives thereof with the enzyme collagenase determines a clear reduction in the enzyme's activity, thus enabling the degradation/removal of the eschar with simultaneous formation of granulation tissue, thanks to the specific action of HA.
Moreover, said polysaccharide protects the healthy tissue surrounding the lesion from the digestive action of collagenase, thus increasing patient compliance with the product.
By slowing down the proteolytic activity of collagenase, hyaluronic acid manifests properties that prove to be the absolute opposite of those of the delivery system described above, as known to an expert in the current state of the art.
A further subject of the present invention is represented by pharmaceutical formulations of a lipophilic nature, containing collagenase in association with HA, that enable the complete stabilisation of the enzyme and, therefore, its maintenance in an active form, at room temperature for prolonged periods of time.